(a) The role of RAGE and its ligand HMGB1 in the regulation of leukocyte integrin activity. Previously we characterized RAGE (receptor for advanced glycation endproducts) as a counter-receptor of the beta2-integrin Mac-1. One of the RAGE ligands, high mobility group B1 (HMGB1) is a nuclear protein, that can be actively secreted by macrophages and dendritic cells upon inflammatory stimuli and is also passively released by necrotic but not apoptotic cells. Extracellular HMGB1 evokes a very strong inflammatory response, thus HMGB1 represents a warning system for tissue injury. Leukocyte recruitment is a prominent hallmark associated with HMGB1-mediated inflammation, however, the underlying mechanisms were not defined yet. Our efforts have defined that HMGB1 has a direct chemotactic activity specifically for neutrophils in vitro and in vivo, which is mediated by the beta2-integrin Mac-1, as HMGB1-mediated neutrophil recruitment was prevented in mice deficient in the beta2-integrin Mac-1 but not LFA-1. In vitro studies indicated that HMGB1 activated the formation of a functional complex in cis on the neutrophil surface between Mac-1 and RAGE, thereby stimulating the avidity and affinity of Mac-1 and Mac-1-dependent neutophil adhesion and migration in a RAGE-dependent manner. These findings were corroborated by bone marrow chimera experiments, which were essential as RAGE is expressed on both neutrophils and endothelial cells. These experiments clearly demonstrated that Mac-1-dependent neutrophil recruitment induced by HMGB1 requires the presence of RAGE on neutrophils but not on endothelial cells. Moreover, HMGB1 induced downstream signalling (NFkappaB activation) in neutrophils required both the presence of Mac-1 and RAGE. (b)Developmentally regulated endothelial locus-1 (Del-1) is an embryonically expressed protein that was previously reported to be produced and secreted by endothelial cells during embryonic development. Interestingly, we found Del-1 to be expressed also in some adult tissues. We found expression of Del-1 in the mouse lung and brain that was strongly upregulated upon inflammatory conditions. We identified a potential interaction between Del-1 and leukocyte integrins. Thus, we postulate that Del-1 may be a specific component of the inflammatory extracellular matrix of the lung and the brain regulating inflammatory cell recruitment. (c) Inside-out signaling regulation of beta2-integrins. The major described pathways of inside out-signaling regulation of integrin activity are the Rap-1-RapL pathway that activates the alpha chain and the talin-pathway linked to the beta chain. These pathways may be regulated by several intracellular pathways including ubiquitin ligases. We found that cbl-b deficiency induces LFA-1 activation. Neutrophil and macrophage recruitment was enhanced by 2-3 fold in cbl-b -/- mice. Additionally, cbl-b-/- macrophages showed increased spreading, adhesion and migration in vitro. Whereas cbl-b-/- cells had no increased Rap1 activity, we found that the interaction between 14-3-3 proteins with the beta2 integrin chain is increased, resulting in higher LFA-1 activation.